Hypothesis: A minimal glycan shield with specific sugar composition on the envelope glycoprotein (Env) of HIV- 1 may be necessary for establishing new infections. In the HIV ?selected? for a new infection, glycan content/composition on Env could differ based on the mode and receptacle of transmission (e.g. rectal, penile, foreskin, vaginal, and cervical tissue in sexual transmission) as well as the cytokine/chemokine milieu and microbiome. Understanding glycan composition on the transmitted HIV-1 in these microenvironments would improve the design and effectiveness of humoral-based HIV-1 vaccine. For the past 15 years, this R01 has compared HIV disease parameters to replicative HIV fitness in T cells, macrophages, dendritic cell cocultures, and various ex vivo, non-mucosal tissues. We have shown that competitive replicative fitness of patient-derived HIV is a surrogate of pathogenesis and in the absence of treatment, was a strong correlate of disease progression. However, our published work indicates that ?pathogenic? and ?transmission? fitness of primary HIV-1 isolates are quite distinct. Using varying conditions with human explant penile and endocervical (EnC) tissues exposed to over 20 different mixtures of HIV-1 derived from acute and chronic disease, we now have a clearer picture of factors impacting HIV transmission. We observed ?trapped? replication of chronic HIV-1 in mucosal (penile and cervical) tissue while the acute HIV-1 isolates were carried through the tissue (possibly via DCs) to infect Th17 or Th1 CD4+ T cells. HIV-1 that ?escapes? mucosal tissues typically have lower affinity for C type lectins and fewer N linked glycosylation sites. To enhance these studies we worked closely with programmers and bioinformatic specialists to develop robust NGS analysis pipelines to determine transmission fitness from multivirus competitions and more recently, we optimized EIS-MS techniques to analyze gp120 glycans. We have proposed four highly inter-related aims to test our hypotheses: Specific Aim 1. In vivo analyses of HIV-1 in endocervical (EnC) and plasma samples and determinants of new HIV transmissions. Examine Env sequence features in the EnC and blood of newly infected women as well as in women with HIV-1 infection in the EnC but who have not seroconverted. Specific Aim 2. Characterizing the properties of transmitted/founder (TF) HIV variant in blood versus the transmitted HIV-1 population in the female and male genital tract: Ex vivo study. Measure trapped replication in the tissue versus transmission in human penile, foreskin, cervical, and vaginal explant model. Specific Aim 3. To measure lectin binding affinity and to analyze the glycosylation pattern on the gp120 Env of highly transmissible TF HIV-1 using new gp120 purification techniques and EIS-MS. Specific Aim 4. To modify the glycans on TF and EnC viruses and to determine optimal HIV Env for male to female HIV transmissions using the same methods described in aims 2 and 3.